Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, deoxynivalenol, and t-2 toxin

ABSTRACT

A time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, deoxynivalenol and T-2 toxin. The kit includes an immunochromatography time-resolved fluorescence test strip and a sample reaction bottle containing freeze-dried products of europium-labeled monoclonal antibodies of toxins, where the immunochromatography time-resolved fluorescence test strip includes a liner, where a water absorption pad, a detection pad and a sample pad are sequentially attached to one side of the liner from top to bottom, adjacent pads are connected in an overlapping manner at a joint, the detection pad uses a nitrocellulose membrane as a base pad, a transverse quality control line and detection lines are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit antimouse polyclonal antibody, the three detection lines are located below the quality control line, and the detection lines each are coated with a toxin-protein conjugate.

BACKGROUND Technical Field

The present disclosure relates to an immunochromatography time-resolvedfluorescence test strip for mycotoxins and pesticides, in particular toa time-resolved fluorescence kit for synchronously detecting compositepollution of 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin, anda preparation method.

Description of Related Art

4,15-diacetoxyscirpenol belongs to trichothecene mycotoxins, and ismainly produced by Fusarium scirpi and Fusarium equiseti.4,15-diacetoxyscirpenol mainly pollutes grains and feed. Its detectionrate in the feed is not higher than that of vomitoxin, its toxicity toanimals is, however, higher than that of vomitoxin. Similar to T-2toxin, 4,15-diacetoxyscirpenol may damage animal hematopoietic organssuch as bone marrow, continuously reduce leucocytes, causecardiomyopathy hemorrhage, and the like. Deoxynivalenol (DON) is alsoreferred to as vomitoxin, can be accumulated in vivo, and has verystrong cytotoxicity, immunotoxicity, embryotoxicity, and teratogeniceffects, causing acute poisoning symptoms such as anorexia, vomiting,diarrhea, fever, unsteadiness and unresponsiveness of humans and animalsand damaging a hematopoietic system to cause death in severe cases. UNFood and Agriculture Organization and World Health Organization havealready determined deoxynivalenol as one of the most dangerous andnaturally occurring food pollutants, and deoxynivalenol is listed inpriorities of international research. T-2 toxin has the strongesttoxicity in trichothecene mycotoxins. The foregoing three toxins allmainly pollute grain crops such as wheat, barley and corn, and productsthereof, causing great harm to human health and animal husbandry. Since4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin always causecomposite pollution to grains, a convenient and rapid detectiontechnology is needed to rapidly and synchronously screen these threetypes of pollutants, so as to meet a regulation requirement forcontrolling toxin pollution in agricultural products.

Currently, detecting methods for these toxins mainly include liquidchromatography, gas chromatography-mass spectrometry, liquidchromatography-mass spectrometry, and the like. These methods are highin stability, sensitivity, and accuracy, but preprocessing steps arecomplex and sample detection costs are high. Immunochromatographyovercomes the foregoing defects. Based on antigen and antibody specificreactions, nitrocellulose is used to fix antigens, free targets in achromatography process compete with the antigens on detection lines tobind to labeled antibodies, and a quantity of the targets contained in asample is calculated by using a quantity of labels bound to thedetection lines. Time-resolved fluorescence immunochromatographic assay(TRFICA) uses europium as a high-affinity probe, has the advantages ofbeing high in sensitivity, stable in property, free from interference offluorescence background, short in detection time, and the like, and isquite suitable for developing a pesticide residue rapid detectionmethod.

Therefore, developing a time-resolved fluorescence kit for synchronouslydetecting composite pollution of 4,15-diacetoxyscirpenol (anguidin),deoxynivalenol, and T-2 toxin is of a great necessity and has quiteimportant significance.

SUMMARY

To solve the problem, the present disclosure provides a time-resolvedfluorescence kit capable of synchronously detecting composite pollutionof 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin, and apreparation method. The time-resolved fluorescence kit can be used tosynchronously detect quantities of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin contained in a sample, and has thecharacteristics of simple and rapid operation and high sensitivity.

To solve the foregoing technical problem, the technical solution adoptedby the present disclosure is as follows:

a time-resolved fluorescence kit for synchronously detecting4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin includes animmunochromatography time-resolved fluorescence test strip and a samplereaction bottle containing freeze-dried products of an europium-labeledanti-4,15-diacetoxyscirpenol monoclonal antibody, an europium-labeledanti-deoxynivalenol monoclonal antibody, and an europium-labeledanti-T-2 toxin monoclonal antibody, where the immunochromatographytime-resolved fluorescence test strip includes a liner, where a waterabsorption pad, a detection pad, and a sample pad are sequentiallyattached to one side of the liner from top to bottom, adjacent pads areconnected in an overlapping manner at a joint, the detection pad uses anitrocellulose membrane as a base pad, a transverse quality control lineand detection lines are arranged on the nitrocellulose membrane from topto bottom, the quality control line is coated with a rabbit antimousepolyclonal antibody, the three detection lines are located below thequality control line, and the detection lines are coated with a4,15-diacetoxyscirpenol-bovine serum albumin conjugate, adeoxynivalenol-bovine serum albumin conjugate, and a T-2 toxin-ovalbuminconjugate respectively; and the anti-4,15-diacetoxyscirpenol monoclonalantibody is secreted by a hybridoma cell strain DAS5G11E7 with thepreservation number of CCTCC NO:C201881 and this hybridoma cell strainDAS5G11E7 has already been preserved on Apr. 3, 2018 in China Center ForType Culture Collection (CCTCC), the preservation address is WuhanUniversity, Wuhan, China, and the preservation number is CCTCC NO.C201881.

According to the foregoing solution, the europium-labeledanti-4,15-diacetoxyscirpenol monoclonal antibody is prepared by usingthe following method: evenly mixing an europium labeling reagent and a4,15-diacetoxyscirpenol monoclonal antibody according to a mass ratio of1:(0.04-0.3), oscillating on a shaking table for 2 h to 4 h,centrifuging to remove supernate, and blocking redundant binding siteson a surface of the europium labeling reagent to obtain the targetproduct, namely the europium-labeled anti-4,15-diacetoxyscirpenolmonoclonal antibody;

the europium-labeled anti-deoxynivalenol monoclonal antibody is preparedby using the following method: evenly mixing an europium labelingreagent and a deoxynivalenol monoclonal antibody according to a massratio of 1:(0.04-0.3), oscillating on a shaking table for 2 h to 4 h,centrifuging to remove supernate, and blocking redundant binding siteson a surface of the europium labeling reagent to obtain the targetproduct, namely the europium-labeled anti-deoxynivalenol monoclonalantibody; and

the europium-labeled anti-T-2 toxin monoclonal antibody is prepared byusing the following method: evenly mixing an europium labeling reagentand a T-2 toxin monoclonal antibody according to a mass ratio of1:(0.04-0.3), oscillating on a shaking table for 2 h to 4 h,centrifuging to remove supernate, and blocking redundant binding siteson a surface of the europium labeling reagent to obtain the targetproduct, namely the europium-labeled anti-T-2 toxin monoclonal antibody.

According to the foregoing solution, the europium labeling reagent isactivated before use, and the activation includes: dissolving theeuropium labeling reagent in a boric acid buffer solution, oscillatingand evenly mixing, adding an EDC solution, oscillating and activatingfor 15 min to 30 min, centrifuging at 10000 rpm to 15000 rpm, adding aboric acid buffer solution, oscillating and evenly mixing, andultrasonically processing.

According to the foregoing solution, a blocking solution for blocking isa boric acid buffer solution that contains 0.5% to 1% of BSA.

According to the foregoing solution, in the immunochromatographytime-resolved fluorescence test strip, the water absorption pad is 15 mmto 35 mm in length and 3 mm to 5 mm in width, the sample pad is 12 mm to18 mm in length and 2 mm to 5 mm in width, and an overlapping length ofadjacent pads is 1 mm to 3 mm; on the detection pad in theimmunochromatography time-resolved fluorescence test strip, a distancebetween the detection line close to the quality control line and anupper edge of the nitrocellulose membrane is 15 mm to 20 mm, a distancebetween every two adjacent detection lines is 1.5 mm to 4.5 mm, and adistance between the detection line close to the quality control lineand the quality control line and detection lines is 4 mm to 10 mm; andthe sample reaction bottle is a 1-5 mL bayonet bottle.

According to the foregoing solution, on the detection pad in theimmunochromatography time-resolved fluorescence test strip, a coatingquantity of the 4,15-diacetoxyscirpenol-bovine serum albumin conjugaterequired for each centimeter of detection line is 0.4 μg to 0.8 μg, acoating quantity of the deoxynivalenol-bovine serum albumin conjugaterequired for each centimeter of detection line is 0.8 μg to 1.0 μg, anda coating quantity of the T-2 toxin-ovalbumin conjugate required foreach centimeter of detection line is 0.8 μg to 1.0 μg; and

a quantity of the freeze-dried product of the europium-labeledanti-4,15-diacetoxyscirpenol monoclonal antibody contained in the samplereaction bottle is 0.1 μg to 0.3 μg, a quantity of the freeze-driedproduct of the europium-labeled anti-deoxynivalenol monoclonal antibodycontained in the sample reaction bottle is 0.2 μg to 0.4 μg, and aquantity of the freeze-dried product of the europium-labeled anti-T-2toxin monoclonal antibody contained in the sample reaction bottle is 0.2μg to 0.4 μg.

According to the foregoing solution, preferably, an IC50 of theanti-deoxynivalenol monoclonal antibody is less than or equal to 15 ppb.

According to the foregoing solution, an IC50 of the anti-T-2 toxinmonoclonal antibody is less than or equal to 2 ppb.

According to the foregoing solution, the time-resolved fluorescence kitfor synchronously detecting composite pollution of4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin further includesa sample diluent, where the sample diluent includes 0.01% to 0.30% byvolume of Tween-20, 0.5% to 1.5% by volume of sucrose, and 0.1% to 1% byvolume of a bovine serum albumin (BSA) aqueous solution by volumepercentage.

According to the foregoing solution, a preparation method for thetime-resolved fluorescence test strip includes the following steps:

(1) cutting water absorption paper into the water absorption pad;

(2) preparation of the detection pad:

preparing coating solutions with a concentration of 0.25 mg/mL to 2mg/mL from the 4,15-diacetoxyscirpenol-bovine serum albumin conjugate,the deoxynivalenol-bovine serum albumin conjugate, and the T-2toxin-ovalbumin conjugate, coating the nitrocellulose membrane with thecoating solutions at intervals in a streak manner to obtain the threedetection lines, respectively, and drying at 37° C. to 40° C. for 30 minto 60 min; where a coating quantity of the4,15-diacetoxyscirpenol-bovine serum albumin conjugate required for eachcentimeter of the detection line coated with the4,15-diacetoxyscirpenol-bovine serum albumin conjugate is 0.2 μg to 0.8μg, a coating quantity of the deoxynivalenol-bovine serum albuminconjugate required for the detection line coated with thedeoxynivalenol-bovine serum albumin conjugate is 0.2 μg to 1.0 μg, and acoating quantity of the T-2 toxin-ovalbumin conjugate required for thedetection line coated with the T-2 toxin-ovalbumin conjugate is 0.2 μgto 1.0 μg;

on the detection pad in the immunochromatography time-resolvedfluorescence test strip, a distance between the detection line close tothe quality control line and an upper edge of the nitrocellulosemembrane is 15 mm to 20 mm, a distance between every two adjacentdetection lines is 1.0 mm to 5.5 mm, and a distance between thedetection line close to the quality control line and the quality controlline is 5 mm to 10 mm; and

preparing a coating solution with a concentration of 0.1 mg/mL to 0.45mg/mL from the rabbit antimouse polyclonal antibody, and transverselycoating the nitrocellulose membrane with the coating solution in aposition 5 mm to 10 mm away from the detection lines in a streak mannerto obtain the quality control line, where a coating quantity of therabbit antimouse polyclonal antibody required for each centimeter of thequality control line is 0.4 μg to 0.8 μg; and then, drying at 37° C. to40° C. for 30 min to 60 min;

(3) preparation of the sample pad:

putting a fiberglass membrane into a blocking solution to be soaked,taking out the soaked fiberglass membrane, and drying at 37° C. to 40°C. for 4 h to 10 h to obtain the sample pad, and then preserving thesample pad in a dryer at room temperature; and

(4) assembling of the immunochromatography time-resolved fluorescencetest strip:

sequentially attaching the water absorption pad, the detection pad, andthe sample pad to one side of a paperboard from top to bottom to obtainthe immunochromatography time-resolved fluorescence test strip, whereadjacent pads are connected in an overlapping manner at a joint, and anoverlapping length is 1 mm to 3 mm.

According to the foregoing solution, during the preparation of theimmunochromatography time-resolved fluorescence test strip, a coatingbuffer solution used to prepare the 4,15-diacetoxyscirpenol-bovine serumalbumin conjugate coating solution, the deoxynivalenol-bovine serumalbumin conjugate coating solution, and the T-2 toxin-ovalbuminconjugate coating solution includes in each 10 mL: 0.1 g of bovine serumalbumin, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g ofsodium phosphate dibasic dodecahydrate, 0.002 g of potassium chloride,and 0.002 g of monopotassium phosphate;

a coating buffer solution used to prepare the rabbit antimousepolyclonal antibody coating solution includes in each 10 mL: 0.002 g ofsodium azide, 0.08 g of sodium chloride, 0.029 g of sodium phosphatedibasic dodecahydrate, 0.002 g of potassium chloride, and 0.002 g ofmonopotassium phosphate; and

the blocking solution used during the preparation of theimmunochromatography time-resolved fluorescence test strip includes ineach 100 mL: 0.5 g to 2 g of ovalbumin, 2 g of sucrose, 0.02 g of sodiumazide, 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasicdodecahydrate, 0.02 g of potassium chloride, and 0.02 g of monopotassiumphosphate.

Application of the foregoing immunochromatography time-resolvedfluorescence fast detection kit to detecting 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin includes: preprocessing a to-be-testedsample to obtain a to-be-tested sample solution, adding the to-be-testedsample solution into a sample reaction bottle, evenly mixing, insertinga time-resolved fluorescence test strip, carrying out a reaction at 37°C. for 6 min, and detecting by using a time-resolved fluorescence testerto obtain a ratio of time-resolved fluorescence intensity of thedetection lines (T) to time-resolved fluorescence intensity of thequality control line (C) on the immunochromatography time-resolvedfluorescence test strip; based on a curve of respective relationshipsbetween the ratio (T/C) of time-resolved fluorescence intensity of thedetection lines to time-resolved fluorescence intensity of the qualitycontrol line on the immunochromatography time-resolved fluorescence teststrip obtained in advance and concentrations of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin, obtaining quantities of4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin contained in theto-be-tested sample solution, and finally converting these quantitiesinto quantities of 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2toxin contained in the to-be-tested sample.

According to the foregoing solution, the curve of respectiverelationships between the ratio (T/C) of time-resolved fluorescenceintensity of the detection lines to time-resolved fluorescence intensityof the quality control line on the immunochromatography time-resolvedfluorescence test strip and concentrations of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin is obtained by using the following method:

(1) preparing 4,15-diacetoxyscirpenol standard solutions in seriesconcentrations, preparing deoxynivalenol standard solutions in seriesconcentrations, and preparing T-2 toxin standard solutions in seriesconcentrations;

(2) adding proper volumes of 4,15-diacetoxyscirpenol, deoxynivalenol,and T-2 toxin standard solutions in each concentration into the samplereaction bottles respectively, evenly mixing, inserting theimmunochromatography time-resolved fluorescence test strips, carryingout a reaction at 37° C. for 10 min, detecting by using a time-resolvedfluorescence immunoassay system to obtain values of time-resolvedfluorescence intensity of detection lines (T) and quality control lines(C) on the immunochromatography time-resolved fluorescence test strips,and then obtaining ratios (T/C) of time-resolved fluorescence intensityof detection lines to time-resolved fluorescence intensity of qualitycontrol lines on the immunochromatography time-resolved fluorescencetest strips; and

(3) obtaining, through fitting, the curve of relationships between theratios (T/C) of time-resolved fluorescence intensity of detection linesto time-resolved fluorescence intensity of quality control lines on theimmunochromatography time-resolved fluorescence test strips and theconcentrations of 4,15-diacetoxyscirpenol, obtaining, through fitting,the curve of relationships between the ratios (T/C) of time-resolvedfluorescence intensity of detection lines to time-resolved fluorescenceintensity of quality control lines on the immunochromatographytime-resolved fluorescence test strips and the concentrations ofdeoxynivalenol, and obtaining, through fitting, the curve ofrelationships between the ratios (T/C) of time-resolved fluorescenceintensity of detection lines to time-resolved fluorescence intensity ofquality control lines on the immunochromatography time-resolvedfluorescence test strips and the concentrations of T-2 toxin.

The present disclosure has the beneficial effects:

(1) 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin are rapidlyand synchronously detected. For the immunochromatography time-resolvedfluorescence kit provided in the present disclosure, the threemycotoxins including 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2toxin can be synchronously and rapidly detected on one test strip.Specifically, the 4,15-diacetoxyscirpenol monoclonal antibody used ishigh in specificity (cross reactivity with T-2 toxin, vomitoxin (DON),or the like each are less than 0.01%), and high in sensitivity, and itcan be ensured that detections of the mycotoxins are free frominterference, simple and rapid.

(2) Sensitivity is high. For the immunochromatography time-resolvedfluorescence kit provided in the present disclosure, a low of detectionof 4,15-diacetoxyscirpenol in a detection solution is 0.5 ng/mL, a lowof detection of deoxynivalenol is 1.0 ng/mL, a low of detection of T-2toxin is 0.1 ng/mL, and these limits of detection can meet requirementsof European Union for limits in food.

(3) A sample preprocessing method is simple. For sample preprocessing,only after adding a methanol water extract to a sample, ultrasonicallyextracting for 5 min to 10 min, standing for 5 min to 10 min, takingsupernate to be allowed to pass through a filtering membrane, anddiluting, detection can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic structural diagram of an immunochromatographytime-resolved fluorescence test strip for 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin provided by the present disclosure; in thefigure: 1 Water absorption pad; 2 Detection pad; 3 Sample pad; 4 Qualitycontrol line; 5 4,15-diacetoxyscirpenol detection line; 6 Deoxynivalenoldetection line; and 7 T-2 toxin detection line;

FIG. 2 illustrates affinity measurement data of a4,15-diacetoxyscirpenol monoclonal antibody provided by the presentdisclosure; and

(a) of FIG. 3 illustrates results of cross reactions between a4,15-diacetoxyscirpenol monoclonal antibody and other mycotoxinsprovided by the present disclosure; and (b) of FIG. 3 illustrates a4,15-diacetoxyscirpenol enzyme linked immunoassay standard curveconstructed by a 4,15-diacetoxyscirpenol monoclonal antibody provided bythe present disclosure.

DESCRIPTION OF THE EMBODIMENTS

Obtaining of anti-4,15-diacetoxyscirpenol monoclonal antibody

An anti-4,15-diacetoxyscirpenol monoclonal antibody was secreted by ahybridoma cell strain DAS5G11E7 with the preservation number of CCTCCNO. C201881. A preparation method included:

injecting the hybridoma cell strain DAS5G11E7 into BALB/c micepreprocessed by using a Freund's incomplete adjuvant, collecting ascitesof the mice, and purifying the antibody by using a caprylicacid-ammonium sulfate method. Specific operation included: filtering theascites of the mice by using double-layer filter paper, centrifuging at4° C. and 12000 r/min for 15 min or above, sucking supernate, mixing theascites supernate with 4 times volume of an acetate buffer solution,slowly adding n-caprylic acid while stirring, with 30 μL to 35 μL ofn-caprylic acid required for each mL of ascites, mixing at roomtemperature for 30 min to 60 min, and standing at 4° C. for 2 h orabove; centrifuging at 12000 r/min and 4° C. for 30 min or above,discarding the obtained precipitate, filtering the resulting supernateby using double-layer filter paper, adding a phosphate buffer solutionwith a molar concentration of 0.1 mol/L and a pH of 7.4, with a volumebeing 1/10 that of the obtained filtrate, adjusting the pH of the mixedsolution to 7.4 by using a 2 mol/L sodium hydroxide solution, slowlyadding ammonium sulfate in an ice bath to reach an ammonium sulfatefinal concentration of 0.277 g/mL, standing at 4° C. for 2 h or above,then centrifuging at 12000 r/min and 4° C. for 30 min or above,discarding supernate, resuspending the resulting precipitate by using aphosphate buffer solution with a molar concentration of 0.01 mol/L and apH of 7.4, with a volume being 1/10 that of the original ascites,filling a dialysis bag, dialyzing by using 0.01 mol/L PBS for 2 d, thendialyzing by using PB for 2 d, taking a protein solution out of thedialysis bag, centrifuging, collecting supernate, discarding theobtained precipitate, prefreezing at −70° C., and putting the prefrozenmaterial into a freeze dryer for freeze-drying; and collectingfreeze-dried powder, namely the purified anti-4,15-diacetoxyscirpenolmonoclonal antibody.

The acetate buffer solution was prepared from 0.29 g of sodium acetate,0.141 mL of acetic acid, and water that was added until a constantvolume of 100 mL; the 0.01 mol/L phosphate buffer solution was preparedfrom 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasicdodecahydrate, 0.02 g of potassium chloride, and 0.02 g of monopotassiumphosphate, and water that was added until a constant volume of 100 mL;and the 0.1 mol/L phosphate buffer solution was prepared from 8 g ofsodium chloride, 2.9 g of sodium phosphate dibasic dodecahydrate, 0.2 gof potassium chloride, 0.2 g of monopotassium phosphate, and water thatwas added until a constant volume of 100 mL.

A subtype of the anti-4,15-diacetoxyscirpenol monoclonal antibodysecreted by the hybridoma cell strain DAS5G11E7 was identified to beIgG2b by using a commercially available subtype identification kit.

It was measured by using a conventional non-competitive enzyme-linkedimmunosorbent assay (ELISA) that a valence of the antibody obtained fromthe mouse ascites by purifying might reach 3.2×10⁵, that was, when theantibody was diluted by 3.2×10⁵ times, the solution measurement resultwas positive. It was measured by using a conventional indirectcompetitive ELISA that its sensitivity to 4,15-diacetoxyscirpenol was3.08 ng/mL. Cross reactivity with other mycotoxins, T-2 toxin, HT-2toxin, vomitoxin, 3-acetyldeoxynivalenol, ochratoxin, and fumonisin eachwere less than 0.01% (Table 1; FIG. 3 ). Specificity of the antibodymight be evaluated by using cross reactivity. The monoclonal antibodyfrom the DAS5G11E7 was measured by using the indirect competitive ELISA.Standard solutions in series concentrations were prepared from DAS, T-2toxin, HT-2 toxin, DON, 3-ACDON, OTA, and FB₁, and added together withan equal volume of antibody into ELISA plates respectively to beincubated for 1 h. Other steps were the same as the indirect competitiveELISA. Concentrations of the foregoing toxin standards served asabscissas, and OD values B/130 under 450 nm measured by a microplatereader served as ordinates, to draw a competitive inhibition curve.Cross reactivity was determined by calculating ratios of an IC50 valueof DAS to an IC50 value of other toxins. The calculation formula was asfollows:

CR %=(IC50DAS/IC50other toxins)×100.

TABLE 1 Cross reaction of DAS5G11E7 with other toxins Name of toxinStructure IC50 Cross reactivity DAS

3.08 100% T-2 toxin

>100,000 <0.01% HT-2 toxin

>100,000 <0.01% DON

>100,000 <0.01% 3-acetyl-DON

>100,000 <0.01% FB₁

>100,000 <0.01% OTA

>100,000 <0.01%

Affinity of the DAS5G11E7 was measured by using the indirectnon-competitive ELISA. The ELISA plates were coated with DAS-OVAaccording to concentrations of 1.0 μg/mL, 0.5 μg/mL, 0.25 μg/mL, and0.125 μg/mL, with 100 μL per pore, at 37° C. for 2 h. After blocking wasperformed for 1 h by using a blocking solution, the antibody diluted byPBS (a dilution factor of 1:2) was added into the ELISA plates. Othersteps were the same as the indirect non-competitive ELISA. MeasuredOD450 values served as ordinates, and log values of antibodyconcentrations (mol/L) served as abscissas, to draw four S-shaped curvesof four concentrations. A maximum OD value on the topmost of eachS-shaped curve was found out, namely ODmax, and an antibodyconcentration corresponding to a 50% ODmax value of each curve was foundout. Affinity constants of the antibody were calculated according to theformula Ka=(n−1)/2(n[Ab’]t−[Ab]t) by grouping any two of the fourconcentrations into a group, where [Ab′]t and [Ab]t were antibodyconcentrations corresponding to two 50% maximum OD values in each group,n was a multiple (including three ratios 1:2, 1:4, and 1:8) of a coatedantigen concentration in each group, and six Ka values were obtained intotal. The six obtained Ka values were averaged to obtain the affinityof an anti-4,15-diacetoxyscirpenol mouse ascites antibody by using theenzyme-linked immunosorbent assay (ELISA), which might reach 5.4×10⁸L/moL (FIG. 2 ).

Screening of hybridoma cell strain DAS5G11E7

1. Animal Immunization

BALB/c mice that were six to seven weeks old were immunized by a4,15-diacetoxyscirpenol complete antigen DAS-BSA prepared in alaboratory. For the first immunization, the 4,15-diacetoxyscirpenolcomplete antigen and an equal volume of Freund's complete adjuvant wereemulsified and subcutaneously injected at multiple points in necks andbacks of the mice. The second immunization was performed four weekslater. A Freund's incomplete adjuvant and an equal volume of4,15-diacetoxyscirpenol complete antigen were emulsified and injected inabdomens of the mice. The interval between the third immunization andthe second immunization was 4 weeks, and the immunization manners werethe same. The fourth immunization was performed 3 weeks after the thirdimmunization, and the immunization manner was the same as that in thesecond immunization, that was, intraperitoneal injection. In the fourimmunizations, dosages were the same, that was, 70 μg for each mouse.For the first three immunizations, eight days to ten days after eachimmunization, blood was sampled from caudal vein, serum was separatedout, and valences of the serums of the mice were measured by using theindirect ELISA. Eight days after the third immunization, tails were cutfor blood sampling, and the mice corresponding to the serums with bothrelatively high valences and sensitivities were selected for the lastenhanced immunization, with an immunization dosage twice the foregoingdosage.

2. Cell Fusion

Three days after the enhanced immunization, PEG with a weight percentageof 50% and a polyethylene glycol molecular weight of 1450 was used as afusogen, and cell fusion was performed by using a conventional method,specifically including the steps: sacrificing the mice by cervicaldislocation under aseptic conditions, taking out spleens, using ahomogenizer to crush the spleens, separating out splenocytes by using afilter screen, mixing the splenocytes and murine myeloma cells SP2/0according to a ratio of 5:1, centrifuging, resuspending mixed cells byusing an RPMI-1640 basal medium, centrifuging, and discarding supernate;and adding 1 mL to 2 mL of 50% PEG within 1 min in total, adding 10 mLto 20 mL of RPMI-1640 basal medium along the wall, centrifuging,discarding supernate, resuspending fused cells at a tube bottom by using20 mL of a cell complete medium containing 1% of HAT, adding suspendedcells to 80 mL of a semi-solid medium, evenly mixing, adding theobtained mixture into a 6-pore cell culture plate, with 1.5 mL per pore,and culturing in a 37° C. carbon dioxide incubator. The cell completemedium containing 1% of HAT included: 20% (volume percentage) of fetalbovine serum, 75% (volume percentage) of RPMI-1640 basal medium, 1%(weight percentage) of L-glutamine, 1% (volume percentage) of HEPES, 1%(volume percentage) of double antibodies (10000 units per milliliter ofpenicillin and 10000 micrograms per milliliter of streptomycin), 2%(volume percentage) of growth factor (HFCS), and 1% (weight percentage)of hypoxanthine-aminopterin-thymidine (HAT) and methylcellulose, whichwas purchased from the sigma-Aldrich company.

Screening and Cloning of Cell Strain

When cell colonies grew to be visible to the naked eye two to threeweeks after the cell fusion, clones were picked from the culture mediumby using a micropipettor and transferred to a 96-pore cell culture plateto be cultured in HAT liquid. When cells grew to ⅔ height from the porebottom, a culture supernate was sucked for detection. A two-stepscreening method was applied. In the first step, the indirect ELISA wasused to screen out positive pores that were resistant to4,15-diacetoxyscirpenol other than a carrier protein BSA. In the secondstep, indirect competitive ELISA was used to detect the positive poresscreened out in the first step, and 4,15-diacetoxyscirpenol was used asa competitor to select the pores with both relatively large lightabsorption value and relatively high sensitivity (relatively large lightabsorption value meant that a final measurement value of a pore with thecompetitor of 0, that was, a positive control hole, was relativelylarge, and relatively high sensitivity meant that a competitorconcentration at an inhibition ratio of 50%, that is, the IC₅₀ value wasrelatively small). Subcloning was performed by using a limiting dilutionmethod. After the subcloning, the same two-step method was used toperform detection. After subcloning was repeatedly performed for 4-5times, the hybridoma cell strain DAS5G11E7 was obtained. This hybridomacell strain has already been preserved on Apr. 3, 2018 in China CenterFor Type Culture Collection (CCTCC), the preservation address is WuhanUniversity, Wuhan, China, and the preservation number is CCTCC NO:C201881.

Measurement of sequences of variable regions ofanti-4,15-diacetoxyscirpenol monoclonal antibody, hybridoma cell strainDAS5G11E7 antibody

(1) Extraction of total RNA: a total RNA extraction kit from the Tiangencompany was used to extract, following the instructions, total RNAcapable of producing the hybridoma cell strain DAS5G11E7;

(2) Synthesis of cDNA: with the total RNA obtained in the step (1) as atemplate and oligo(dT)15 as primers, reverse transcription wasperformed, following the SuperScript™-2II reverse transcriptaseinstructions, to synthesize a first strand of cDNA; where the primersoligo(dT)15 were purchased from Invitrogen; and

(3) Cloning of variable region genes by a PCR method: based on conservedsites of mouse antibody gene sequences in GENBANK, primers weredesigned, and the cDNA was used as a template to amplify the antibodyheavy chain and light chain variable region genes. The PCR procedureincluded: at 94° C. for 30 s, at 58° C. for 45 s, at 72° C. for 1 min,amplification for 30 cycles, and finally elongation at 72° C. for 10min. After electrophoretic separation was performed on the PCR productby using 1% (weight percentage) of agarose gel, DNA segments werepurified and recovered by using a kit and connected into a vectorpMD18-T, Escherichia coli DH5a competent cells were transformed,positive clones were picked and sent to Shanghai Sunny BiotechnologyCo., Ltd. for sequencing. For sequences of the primers, the sequences ofthe heavy chain variable region primers were 5′-CAG GTS MAR CTG MAG GAGTCW G-3′(22mer) and 5′-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3′(28mer),where S, M, R, and W were degenerate bases, M=A/C, R=A/G, S=G/C, andW=A/T; and the sequences of the light chain variable region primers were5′-GAC ATC AAG ATG ACC CAG TCT CCA-3′(24mer) and 5′-CCG TTT TAT TTC CAGCTT GGT CCC-3′(24mer).

Resulting gene sequence results: the sequence of a heavy chain variableregion coding gene was 351 bp in length and was shown as SEQ ID NO:1,and it was deduced that a heavy chain variable region encoded by thegene sequence was composed of 117 amino acids and was shown as SEQ IDNO:3 based on the obtained gene sequence. The sequence of a light chainvariable region coding gene was 324 bp in length and was shown as SEQ IDNO:2, and it was deduced that a light chain variable region encoded bythe gene sequence was composed of 108 amino acids and was shown as SEQID NO:4 based on the obtained gene sequence.

The anti-deoxynivalenol monoclonal antibody was preferably theanti-deoxynivalenol monoclonal antibody with an IC50 less than or equalto 15 ppb, for example, the anti-deoxynivalenol monoclonal antibodyproduced by Shandong Lvdu Bio-Sciences&Technology Co., Ltd. In thisembodiment, the anti-deoxynivalenol monoclonal antibody from ShandongLvdu Bio-Sciences&Technology Co., Ltd. was specifically used, and asensitivity IC50 was 12 ppb.

The anti-T-2 toxin monoclonal antibody was preferably the anti-T-2 toxinmonoclonal antibody with an IC50 less than or equal to 2 ppb (2 ng/mL),for example, the anti-T-2 toxin monoclonal antibody produced by ShandongLvdu Bio-Sciences&Technology Co., Ltd. In this embodiment, the anti-T-2toxin monoclonal antibody from Shandong Lvdu Bio-Sciences&TechnologyCo., Ltd. was specifically used, and a sensitivity IC50 was 0.8 ng/mLthrough detection.

Embodiment 4 Time-resolved fluorescence kit for synchronously detecting4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin

A time-resolved fluorescence kit for quantitatively detecting4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin includes afluorescence test strip, a reaction bottle containing aneuropium-labeled anti-4,15-diacetoxyscirpenol monoclonal antibody, aneuropium-labeled anti-deoxynivalenol monoclonal antibody, and aneuropium-labeled anti-T-2 toxin monoclonal antibody, and a samplediluent. The fluorescence test strip includes a paperboard, where awater absorption pad, a detection pad, and a sample pad are sequentiallyattached to one side of the paperboard from top to bottom; adjacent padsare connected in an overlapping manner at a joint, and an overlappinglength is 1 mm; in the immunochromatography time-resolved fluorescencetest strip, the water absorption pad is 38 mm in length and 4 mm inwidth, the detection pad is 25 mm in length and 4 mm in width, thesample pad is 15 mm in length and 4 mm in width, and an overlappinglength of adjacent pads is 1 mm; and the detection pad uses anitrocellulose membrane as a base pad, a transverse quality controlline, a detection line 1, a detection line 2, and a detection line 3 arearranged on the nitrocellulose membrane from top to bottom, and thequality control line is coated with a rabbit antimouse polyclonalantibody.

Obtaining of the fluorescence test strip:

(1) Preparation of the water absorption pad

Water absorption paper was cut into the water absorption pad with aspecification of 18 mm in length and 4 mm in width;

(2) Preparation of the detection pad

Coating of detection lines:

A coating solution with a concentration of 1 mg/mL was prepared from a4,15-diacetoxyscirpenol coating antigen by using a coating buffersolution. A nitrocellulose membrane was transversely coated with thecoating solution in a streak manner in a position 12 mm away from anupper edge of the nitrocellulose membrane to obtain a detection line,where a coating quantity of the coating antigen required for eachcentimeter of the detection line was 0.6 μg. Then, drying was performedat 37° C. for 60 min.

A coating solution with a concentration of 0.25 mg/mL was prepared froma deoxynivalenol coating antigen by using a coating buffer solution. Thenitrocellulose membrane was transversely coated with the coatingsolution in a streak manner in a position 8 mm away from the upper edgeof the nitrocellulose membrane to obtain a detection line, where acoating quantity of the coating antigen required for each centimeter ofthe detection line was 0.4 μg. Then, drying was performed at 37° C. for60 min.

A coating solution with a concentration of 0.25 mg/mL was prepared froma T-2 toxin coating antigen by using a coating buffer solution. Thenitrocellulose membrane was transversely coated with the coatingsolution in a streak manner in a position 8 mm away from the upper edgeof the nitrocellulose membrane to obtain a detection line, where acoating quantity of the coating antigen required for each centimeter ofthe detection line was 0.4 μg. Then, drying was performed at 37° C. for60 min.

The coating buffer solution included in each 10 mL: 0.1 g of bovineserum albumin (BSA), 0.002 g of sodium azide, 0.08 g of sodium chloride,0.029 g of sodium phosphate dibasic dodecahydrate, 0.002 g of potassiumchloride, and 0.002 g of monopotassium phosphate.

Coating of the quality control line:

A coating solution with a concentration of 0.25 mg/mL was prepared froma rabbit antimouse polyclonal antibody by using a coating buffersolution. The nitrocellulose membrane was transversely coated with thecoating solution in a position 4 mm away from the detection lines in astreak manner to obtain the quality control line, where a coatingquantity of the rabbit antimouse polyclonal antibody required for eachcentimeter of the quality control line was 0.4 μg. Then, drying wasperformed at 37° C. for 2 h.

The coating buffer solution included in each 10 mL:

0.1 g of bovine serum albumin, 0.002 g of sodium azide, 0.08 g of sodiumchloride, 0.029 g of sodium phosphate dibasic dodecahydrate, 0.002 g ofpotassium chloride, and 0.002 g of monopotassium phosphate.

The nitrocellulose membrane was 25 mm in length and 4 mm in width.

(3) Preparation of the sample pad:

A fiberglass membrane was cut to a specification of 15 mm in length and4 mm in width, put into a blocking solution to be soaked, taken out, anddried at 37° C. for 6 h to obtain the sample pad. Then, the sample padwas preserved in a dryer at room temperature.

The blocking solution was prepared from 2.9 g of sodium phosphatedibasic dodecahydrate, 0.3 g of sodium dihydrogen phosphate dihydrate,1.0 g of Tween-20, 1.0 g of polyvinyl pyrrolidone (PVPK-30), 0.25 g ofEDTA, 0.5 g of bovine serum albumin (BSA), 0.02 g of sodium azide, andwater that was added until a constant volume of 100 mL.

(4) Assembling of the fluorescence test strip:

The water absorption pad, the detection pad, and the sample pad weresequentially attached to one side of a paperboard from top to bottom toobtain the fluorescence test strip, where adjacent pads were connectedin an overlapping manner at a joint, and an overlapping length was 1 mm.

Obtaining of the europium-labeled anti-4,15-diacetoxyscirpenolmonoclonal antibody:

200 μL of an europium labeling reagent (a particle size of 100 nm, and asolid content of 1%) was added into 800 μL of a 0.2 mol/L boric acidbuffer solution with a pH of 8.18. Oscillating and even mixing wereperformed. Ultrasonic processing was performed for 3 s. 40 μL of a 15mg/mL EDC solution was added. Oscillating and even mixing were performedfor 15 min. Centrifuging was performed at 13000 r/min and 10° C. for 10min to remove supernate. For redissolving, 1 mL of a boric acid buffersolution was added. Oscillating and even mixing were performed.Ultrasonic processing was performed for 3 s. 20 μg of a4,15-diacetoxyscirpenol monoclonal antibody was added to be evenlymixed. Processing was performed on a shaking table overnight at 250r/min and 25° C. Centrifuging was performed again to remove supernate.For redissolving, 1 mL of a boric acid buffer solution that contains0.5% of BSA was added. Oscillating and even mixing were performed.Ultrasonic processing was performed for 10 min. Processing was performedon a shaking table at 25° C. for 2 h to obtain a target product, namelythe europium-labeled anti-4,15-diacetoxyscirpenol monoclonal antibody.The europium labeling reagent might be purchased from Shanghai YouniBiotechnology Co., Ltd., but was not limited thereto.

Obtaining of the europium-labeled anti-deoxynivalenol monoclonalantibody:

200 μL of an europium labeling reagent (a particle size of 100 nm, and asolid content of 1%) was added into 800 μL of a 0.2 mol/L boric acidbuffer solution with a pH of 8.18. Oscillating and even mixing wereperformed. Ultrasonic processing was performed for 3 s. 40 of a 15 mg/mLEDC solution was added. Oscillating and even mixing were performed for15 min. Centrifuging was performed at 13000 r/min and 10° C. for 10 minto remove supernate. For redissolving, 1 mL of a boric acid buffersolution was added. Oscillating and even mixing were performed.Ultrasonic processing was performed for 3 s. 20 μg of a deoxynivalenolmonoclonal antibody was added to be evenly mixed. Processing wasperformed on a shaking table overnight at 250 r/min and 25° C.Centrifuging was performed again to remove supernate. For redissolving,1 mL of a boric acid buffer solution that contains 0.5% of BSA wasadded. Oscillating and even mixing were performed. Ultrasonic processingwas performed for 10 min. Processing was performed on a shaking table at25° C. for 2 h to obtain a target product, namely the europium-labeledanti-deoxynivalenol monoclonal antibody. The europium labeling reagentmight be purchased from Shanghai Youni Biotechnology Co., Ltd., but wasnot limited thereto.

Obtaining of the europium-labeled anti-T-2 toxin monoclonal antibody:

200 μL of an europium labeling reagent (a particle size of 100 nm, and asolid content of 1%) was added into 800 μL of a 0.2 mol/L boric acidbuffer solution with a pH of 8.18. Oscillating and even mixing wereperformed. Ultrasonic processing was performed for 3 s. 40 μL of a 15mg/mL EDC solution was added. Oscillating and even mixing were performedfor 15 min. Centrifuging was performed at 13000 r/min and 10° C. for 10min to remove supernate. For redissolving, 1 mL of a boric acid buffersolution was added. Oscillating and even mixing were performed.Ultrasonic processing was performed for 3 s. 20 μg of a T-2 toxinmonoclonal antibody was added to be evenly mixed. Processing wasperformed on a shaking table overnight at 250 r/min and 25° C.Centrifuging was performed again to remove supernate. For redissolving,1 mL of a boric acid buffer solution that contains 0.5% of BSA wasadded. Oscillating and even mixing were performed. Ultrasonic processingwas performed for 10 min. Processing was performed on a shaking table at25° C. for 2 h to obtain a target product, namely the europium-labeledanti-T-2 toxin monoclonal antibody. The europium labeling reagent mightbe purchased from Shanghai Youni Biotechnology Co., Ltd., but was notlimited thereto.

Application of the immunochromatography time-resolved fluorescence teststrip to quantitatively detecting 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin:

1. Construction of a curve of relationships between the ratio (T/C) oftime-resolved fluorescence intensity of the detection lines totime-resolved fluorescence intensity of the quality control line on thefluorescence test strip and concentrations of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin:

(1) Sample preprocessing was performed on grains that were determined tobe negative on 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin byusing high performance liquid chromatography (HPLC). To 25 g of a grainsample, 100 mL of a 80% methanol aqueous solution was added.Homogenizing and extracting were performed for 5 min. The extract wasallowed to stand. The extract after standing was allowed to pass throughdouble-layer filter paper. Filtrate was collected and diluted accordingto a ratio of 1:4.

(2) Labeling of standard substances was performed on the grain samplediluent to prepare a mixed standard solution. 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin were added according to a ratio of 1:1:1to prepare the mixed standard solution. Concentrations of standards weresequentially 1.5 ng/mL, 0.5 ng/mL, 0.15 ng/mL, 0.05 ng/mL, 0.015 ng/mL,and 0.005 ng/mL. Concentrations of 4,15-diacetoxyscirpenol standardsolutions were 15 ng/mL, 5 ng/mL, 1.5 ng/mL, 0.5 ng/mL, 0.15 ng/mL, and0.05 ng/mL respectively.

(3) Into a sample bottle, 200 μL of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin mixed standard solution was taken to beadded. The solution was added into a sample reaction bottle containingeuropium-labeled monoclonal antibodies to be evenly mixed. One end ofthe sample pad of the fluorescence test strip was inserted into thereaction bottle for a reaction at 37° C. for 10 min. The product wasloaded onto a machine to be detected. The detecting instrument was atime-resolved fluorescence immunoassay system and had an excitationwavelength of 365 nm and an emission wavelength of 615 nm. Ratios (T/C)of time-resolved fluorescence intensity of detection lines totime-resolved fluorescence intensity of quality control lines on thefluorescence test strips were detected.

(4) Concentrations of 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2toxin standards served as abscissas, and ratios (T/C) of time-resolvedfluorescence intensity of detection lines to time-resolved fluorescenceintensity of quality control lines corresponding to standard solutionswith various concentrations served as ordinates, to obtain the curve ofrelationships through fitting. Effective detection ranges of the methodincluded: a range of 0.5 ng/mL to 20 ng/mL for 4,15-diacetoxyscirpenol,a range of 1.0 ng/mL to 25 ng/mL for deoxynivalenol, and a range of 0.1ng/mL to 10.5 ng/mL for T-2 toxin.

2. Spike and recovery test for quantities of 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin contained in wheat samples:

To 20 g of wheat samples, 100 mL of a 80% methanol aqueous solution wasadded. Homogenizing and extracting were performed for 5 min. The extractwas allowed to stand. The extract after standing was allowed to passthrough double-layer filter paper. Filtrate was collected and dilutedaccording to a ratio of 1:1.4,15-diacetoxyscirpenol (deoxynivalenol, andT-2 toxin) standards with concentrations of 1 ng/mL, 5 ng/mL, and 10ng/mL were accurately added thereto respectively. Into a sample reactionbottle containing europium-labeled monoclonal antibodies, 200 μL of ato-be-tested sample detection solution was added to be evenly mixed. Oneend of the sample pad of the fluorescence test strip was inserted intothe reaction bottle for a reaction at 37° C. for 10 min. The product wasloaded onto a machine to be detected. The detecting instrument was atime-resolved fluorescence immunoassay system and had an excitationwavelength of 365 nm and an emission wavelength of 615 nm. Ratios (T/C)of time-resolved fluorescence intensity of detection lines totime-resolved fluorescence intensity of quality control lines on thefluorescence test strips were detected. Then, the ratios weresubstituted into a curve of relationships between the obtained ratios oftime-resolved fluorescence intensity of detection lines to time-resolvedfluorescence intensity of quality control lines on the fluorescence teststrips and concentrations of standard substances, to obtainconcentrations of substances including 4,15-diacetoxyscirpenol,deoxynivalenol, and T-2 toxin in the sample solution. Then, quantitiesof substances including 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2toxin contained in the sample might be obtained based on dilutionratios. It was measured that, in the wheat samples, spike and recoveryrates of 4,15-diacetoxyscirpenol sequentially were 101.0%, 94.5%, and90.1%; spike and recovery rates of deoxynivalenol sequentially were98.9%, 95.5%, and 89.2%; and spike and recovery rates of T-2 toxinsequentially were 108.4%, 97.5%, and 93.0%.

1. A time-resolved fluorescence kit for synchronously detecting4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin, comprising animmunochromatography time-resolved fluorescence test strip and a samplereaction bottle containing freeze-dried products of an europium-labeledanti-4,15-diacetoxyscirpenol monoclonal antibody, an europium-labeledanti-deoxynivalenol monoclonal antibody and an europium-labeled anti-T-2toxin monoclonal antibody, wherein the immunochromatographytime-resolved fluorescence test strip comprises a liner, wherein a waterabsorption pad, a detection pad and a sample pad are sequentiallyattached to one side of the liner from top to bottom, adjacent pads areconnected in an overlapping manner at a joint, the detection pad uses anitrocellulose membrane as a base pad, a transverse quality control lineand detection lines are arranged on the nitrocellulose membrane from topto bottom, the quality control line is coated with a rabbit antimousepolyclonal antibody, the three detection lines are located below thequality control line, and the detection lines are coated with a4,15-diacetoxyscirpenol-bovine serum albumin conjugate, adeoxynivalenol-bovine serum albumin conjugate and a T-2 toxin-ovalbuminconjugate respectively; and the anti-4,15-diacetoxyscirpenol monoclonalantibody is secreted by a hybridoma cell strain DAS5G11E7 with thepreservation number of CCTCC NO: C201881.
 2. The time-resolvedfluorescence kit according to claim 1, wherein the europium-labeledanti-4,15-diacetoxyscirpenol monoclonal antibody is prepared by usingthe following method: evenly mixing an europium labeling reagent and a4,15-diacetoxyscirpenol monoclonal antibody according to a mass ratio of1:0.04-0.3, oscillating on a shaking table for 2 h to 4 h, centrifugingto remove supernate, and blocking redundant binding sites on a surfaceof the europium labeling reagent to obtain the target product, namelythe europium-labeled anti-4,15-diacetoxyscirpenol monoclonal antibody;the europium-labeled anti-deoxynivalenol monoclonal antibody is preparedby using the following method: evenly mixing an europium labelingreagent and a deoxynivalenol monoclonal antibody according to a massratio of 1:0.04-0.3, oscillating on a shaking table for 2 h to 4 h,centrifuging to remove supernate, and blocking redundant binding siteson a surface of the europium labeling reagent to obtain the targetproduct, namely the europium-labeled anti-deoxynivalenol monoclonalantibody; and the europium-labeled anti-T-2 toxin monoclonal antibody isprepared by using the following method: evenly mixing an europiumlabeling reagent and a T-2 toxin monoclonal antibody according to a massratio of 1:0.04-0.3, oscillating on a shaking table for 2 h to 4 h,centrifuging to remove supernate, and blocking redundant binding siteson a surface of the europium labeling reagent to obtain the targetproduct, namely the europium-labeled anti-T-2 toxin monoclonal antibody.3. The time-resolved fluorescence kit according to claim 2, wherein theeuropium labeling reagent is activated before use, and the activationcomprises: dissolving the europium labeling reagent in a boric acidbuffer solution, oscillating and evenly mixing, adding an EDC solution,oscillating and activating for 15 min to 30 min, centrifuging at 10000rpm to 15000 rpm, adding a boric acid buffer solution, oscillating andevenly mixing, and ultrasonically processing; wherein a blockingsolution for blocking is a boric acid buffer solution that contains 0.5%to 1% of BSA.
 4. The time-resolved fluorescence kit according to claim1, wherein in the immunochromatography time-resolved fluorescence teststrip, the water absorption pad is 15 mm to 35 mm in length and 3 mm to5 mm in width, the sample pad is 12 mm to 18 mm in length and 2 mm to 5mm in width, and an overlapping length of adjacent pads is 1 mm to 3 mm;on the detection pad in the immunochromatography time-resolvedfluorescence test strip, a distance between the detection line close tothe quality control line and an upper edge of the nitrocellulosemembrane is 15 mm to 20 mm, a distance between every two adjacentdetection lines is 1.5 mm to 4.5 mm, and a distance between thedetection line close to the quality control line and the quality controlline is 4 mm to 10 mm; and the sample reaction bottle is a 1-5 mLbayonet bottle.
 5. The time-resolved fluorescence kit according to claim1, wherein on the detection pad in the immunochromatographytime-resolved fluorescence test strip, a coating quantity of the4,15-diacetoxyscirpenol-bovine serum albumin conjugate required for eachcentimeter of detection line is 0.4 μg to 0.8 μg, a coating quantity ofthe deoxynivalenol bovine serum albumin conjugate required for eachcentimeter of detection line is 0.8 μg to 1.0 μg, and a coating quantityof the T-2 toxin-ovalbumin conjugate required for each centimeter ofdetection line is 0.8 μg to 1.0 μg; and a quantity of the freeze-driedproduct of the europium-labeled anti-4,15-diacetoxyscirpenol monoclonalantibody contained in the sample reaction bottle is 0.1 μg to 0.3 μg, aquantity of the freeze-dried product of the europium-labeledanti-deoxynivalenol monoclonal antibody contained in the sample reactionbottle is 0.2 μg to 0.4 μg, and a quantity of the freeze-dried productof the europium-labeled anti-T-2 toxin monoclonal antibody contained inthe sample reaction bottle is 0.2 μg to 0.4 μg.
 6. The time-resolvedfluorescence kit according to claim 1, wherein an IC50 of theanti-deoxynivalenol monoclonal antibody is less than or equal to 15 ppb;and an IC50 of the anti-T-2 toxin monoclonal antibody is less than orequal to 2 ppb.
 7. The time-resolved fluorescence kit according to claim1, further comprising a sample diluent, wherein the sample diluentcomprises 0.01% to 0.30% by volume of Tween-20, 0.5% to 1.5% by volumeof sucrose, and 0.1% to 1% by volume of a bovine serum albumin aqueoussolution.
 8. The time-resolved fluorescence kit according to claim 1,wherein a preparation method for the time-resolved fluorescence teststrip comprises the following steps: (1) cutting water absorption paperinto the water absorption pad; (2) preparation of the detection pad:preparing coating solutions with a concentration of 0.25 mg/mL to 2mg/mL from the 4,15-diacetoxyscirpenol-bovine serum albumin conjugate,the deoxynivalenol-bovine serum albumin conjugate and the T-2toxin-ovalbumin conjugate, coating the nitrocellulose membrane with thecoating solutions at intervals in a streak manner to obtain the threedetection lines, respectively, and drying at 37° C. to 40° C. for 30 minto 60 min; wherein a coating quantity of the4,15-diacetoxyscirpenol-bovine serum albumin conjugate required for eachcentimeter of the detection line coated with the4,15-diacetoxyscirpenol-bovine serum albumin conjugate is 0.2 μg to 0.8μg, a coating quantity of the deoxynivalenol bovine serum albuminconjugate required for each centimeter of the detection line coated withthe deoxynivalenol-bovine serum albumin conjugate is 0.2 μg to 1.0 μg,and a coating quantity of the T-2 toxin-ovalbumin conjugate required foreach centimeter of the detection line coated with the T-2toxin-ovalbumin conjugate is 0.2 μg to 1.0 μg; on the detection pad inthe immunochromatography time-resolved fluorescence test strip, adistance between the detection line close to the quality control lineand an upper edge of the nitrocellulose membrane is 15 mm to 20 mm, adistance between every two adjacent detection lines is 1.5 mm to 4.5 mm,and a distance between the detection line close to the quality controlline and the quality control line is 4 mm to 10 mm; and preparing acoating solution with a concentration of 0.1 mg/mL to 0.45 mg/mL fromthe rabbit antimouse polyclonal antibody, and transversely coating thenitrocellulose membrane with the coating solution in a position 4 mm to10 mm away from the detection lines in a streak manner to obtain thequality control line, wherein a coating quantity of the rabbit antimousepolyclonal antibody required for each centimeter of the quality controlline is 0.4 μg to 0.8 μg; and then, drying at 37° C. to 40° C. for 30min to 60 min; (3) preparation of the sample pad: putting a fiberglassmembrane into a blocking solution to be soaked, taking out, and dryingat 37° C. to 40° C. for 4 h to 10 h to obtain the sample pad, and thenpreserving in a dryer at room temperature; and (4) assembling of theimmunochromatography time-resolved fluorescence test strip: sequentiallyattaching the water absorption pad, the detection pad and the sample padto one side of a paperboard from top to bottom to obtain theimmunochromatography time-resolved fluorescence test strip, whereinadjacent pads are connected in an overlapping manner at a joint, and anoverlapping length is 1 mm to 3 mm.
 9. The time-resolved fluorescencekit according to claim 8, wherein during the preparation of theimmunochromatography time-resolved fluorescence test strip, a coatingbuffer solution used to prepare the 4,15-diacetoxyscirpenol-bovine serumalbumin conjugate coating solution, the deoxynivalenol-bovine serumalbumin conjugate coating solution and the T-2 toxin-ovalbumin conjugatecoating solution comprises in each 10 mL: 0.1 g of bovine serum albumin,0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of sodiumphosphate dibasic dodecahydrate, 0.002 g of potassium chloride and 0.002g of monopotassium phosphate; a coating buffer solution used to preparethe rabbit antimouse polyclonal antibody coating solution comprises ineach 10 mL: 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 gof sodium phosphate dibasic dodecahydrate, 0.002 g of potassium chlorideand 0.002 g of monopotassium phosphate; and the blocking solution usedduring the preparation of the immunochromatography time-resolvedfluorescence test strip comprises in each 100 mL: 0.5 g to 2 g ofovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodiumchloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g ofpotassium chloride and 0.02 g of monopotassium phosphate. 10.Application of the time-resolved fluorescence kit according to claim 1to detecting 4,15-diacetoxyscirpenol, deoxynivalenol and T-2 toxin,comprising: preprocessing a to-be-tested sample to obtain a to-be-testedsample solution, adding the to-be-tested sample solution into a samplereaction bottle, evenly mixing, inserting a time-resolved fluorescencetest strip, carrying out a reaction at 37° C. for 6 min, and detectingby using a time-resolved fluorescence tester to obtain a ratio oftime-resolved fluorescence intensity of the detection lines totime-resolved fluorescence intensity of the quality control line on theimmunochromatography time-resolved fluorescence test strip; based on acurve of respective relationships between the ratio of time-resolvedfluorescence intensity of the detection lines to time-resolvedfluorescence intensity of the quality control line on theimmunochromatography time-resolved fluorescence test strip obtained inadvance and concentrations of 4,15-diacetoxyscirpenol, deoxynivalenoland T-2 toxin, obtaining quantities of 4,15-diacetoxyscirpenol,deoxynivalenol and T-2 toxin contained in the to-be-tested samplesolution, and finally converting into quantities of4,15-diacetoxyscirpenol, deoxynivalenol and T-2 toxin contained in theto-be-tested sample.